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Software Manual MO.Affinity Analysis
Software Manual MO.Affinity Analysis
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Contents
1. System Requirements
2. Term Definitions
3. General Layout
4. Saving and Exporting Data
5. Analysis Setup in the Home Screen
6. Data Selection
7. Dose Response Fit
8. Compare Results
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The MO.Affinity Analysis software allows straightforward analysis and evaluation of MicroScale
Thermophoresis data. It allows quantification of binding parameters such as dissociation constants
(Kd) or EC50 values and easy comparison of results e.g. for one target protein binding to different
compounds.
The MO.Affinity Analysis software guides the user through all important steps from data selection to
evaluation by using a clearly organized submenu layout in the task bar. Creating an analysis file will
retain the chosen settings for data analysis. Additionally, the software allows inspection and
exporting of both raw and processed data at any step during data analysis.
This manual explains the main functions integrated in the MO.Affinity Analysis software.
1. System Requirements
If the necessary licenses have been purchased, MO.Affinity Analysis software can be installed on
computers meeting the following requirements:
Operating system: Windows 7/10 Professional 64 bit
CPU: Intel Core i5 or better
RAM: 8 GB or more
Hard disk: 20 GB or more free disk space available
Display resolution: 1600 x 900 or better
Software: Microsoft .NET 4.5.1 framework (included in installer of
MO.Affinity Analysis software)
Operating system language: English or German
An external computer mouse is necessary to access all software features.
2. Term Definitions
Target: The fluorescent molecule. The concentration of the target molecule is constant throughout
a dilution series.
Ligand: Non-fluorescent binding partner. The ligand concentration is varied by serial dilution.
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MST Trace: MST fluorescence signal over time. A typical MST trace contains an initial detection of the sample fluorescence (by default recorded for 3-5 seconds), followed by activation of the MST power to induce the temperature gradient and subsequent detection of thermophoretic changes in fluorescence (by default recorded for 20-30 seconds). Finally, MST power is deactivated and back diffusion of fluorescent molecules is monitored (recorded for a short period only).
MST Run: A run includes a series of MST traces,
typically of a fluorescent target molecule versus a
serial dilution of a ligand.
Merge Set: A series of replicates of MST runs, with
identical MST power, LED/excitation power as well
as target concentrations. Data within one Merge Set
will be averaged and error bars will be calculated
and displayed. Note that the ligand concentrations
do not necessarily have to be identical and can vary
between merged MST runs.
Analysis Set: A complete dataset consisting of a number of Merge Sets or single MST runs, for
parallel analysis and direct comparison. Dose response curves of runs and Merge Sets in Analysis
Sets can be compared in the same charts with the MO.Affinity Analysis software. All runs contained
in one Analysis Set are analyzed with the same evaluation parameters.
Analysis (file): A single Analysis Set, or a collection of Analysis Sets. The analysis can be saved at
any time. An analysis file can be used to integrate a larger number of MST experiments for a
comprehensive and systematic data analysis.
Raw Data: All fluorescence data recorded by the Monolith instrument: MST traces, capillary scans
and shapes, initial fluorescence values and bleaching rates. All raw data can be viewed in the Raw
Data Inspection tool (see Figure 1 and section 3, point 6).
Please note that the capillary scan displayed is the scan recorded before the MST measurement,
not afterwards (applicable to MST measurements performed with MO.Control software).
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Figure 1: MST Raw Data Inspection: (A) Properties, (B) MST Traces, (C) Capillary Scan, (D) Capillary Shape, (E) Initial Fluorescence, (F) Bleaching Rate. Please note that the capillary scan displayed is the scan recorded before the MST measurement, not afterwards (applicable to MST measurements performed with MO.Control software).
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3. General Layout
All major functions of the MO.Affinity Analysis software are organized in the task bar:
Four tabs guide the user through the process of MST data analysis:
1. Home 2. Data Selection 3. Dose Response Fit 4. Compare Results
It is recommended to complete all four steps in this order to ensure proper documentation and
analysis of MST experiments. Movement between tabs during the analysis process is possible, e.g.
to add additional files, edit names of Analysis sets, etc.
Additional buttons in the task bar are:
5. Quick saving of the analysis file.
6. Raw Data Inspection is available at any time during the analysis process by selecting the Raw Data Inspection button on the top right of the window. This will open a separate window which displays all experiment-associated settings and meta-data, as well as detailed charts of raw MST traces, capillary scans, overlays of capillary shapes, initial fluorescence values and bleaching rates. Selected runs and traces will be highlighted in both, the MO.Affinity Analysis main window as well as in the Raw Data Inspection window. For detailed views of Raw Data Inspection options, see Figure 1.
7. Alerts will be displayed on the top right of the main window. Alerts include experimental inconsistencies as well as warnings about potential inconsistencies during data processing and fitting.
Context-related supporting information, such as term definitions and equations, can be found
when clicking the buttons located on each page.
Anything you do in the software can be undone by pressing Ctrl + Z.
4. Saving and Exporting Data
The MO.Affinity Analysis software allows for saving the current analysis at any time, using the drop-
down menu in the top left (click ), the quick save button in the task bar or navigation back to
the Home tab.
Moreover, chart and tabular data can be exported where indicated using the export buttons (click
), which are located in the Dose Response Fit and Compare Results submenus as well as
in the Raw Data Inspection section. Available image formats for export are .svg, .pdf and .png. Note
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that .svg and .pdf contain vector graphics which can be processed by graphic editing software.
Tabular data are saved in .xlsx or .csv format. Results can also be saved as a condensed report in
.pdf format on the Dose Response Fit and Compare Results screens.
5. Analysis Setup in the Home Screen
In the Home screen, create a new MST analysis or load a preexisting analysis file. Analysis files are
saved in the .nta format. Changes in an analysis can be saved at any time.
When creating a new analysis, enter an analysis name and optionally add comments, e.g. purpose
of the analysis, assay conditions etc. Recently opened analysis files are listed chronologically. Start
adding raw data to your analysis here, or in the next tab Data Selection.
6. Data Selection
See Figure 2 for a summary of the options you have for Data Selection.
To add MST runs to the analysis, use the drag-and-drop function for .ntp, .ntdb or .moc files, or use
the load function to browse folders and select single or multiple files. MST runs of the selected file
will appear in the Data Selection window as thumbnails of normalized MST Traces with a description
of name, experiment settings and date. By changing the View option in the top panel, you can alter
the presentation of the MST data thumbnails to Dose Response, Capillary Shape or Initial
Fluorescence.
Before data analysis is performed, choose the type of analysis. To
analyze binding by MST, click on the MST button in the Choose Analysis
Type panel. In cases of ligand-induced fluorescence changes where the
fluorescence values of each capillary are used to determine binding
constants, click the Initial Fluorescence button.
Note: Use the Initial Fluorescence analysis if there is a ligand-concentration dependent change in sample fluorescence >±20 %. Refer to the User Starting Guide or the MO.Control software for more information.
Note: The data points presented in the “Dose Response” thumbnail view
correspond to the Fnorm values determined after the MST power dependent time intervals (see section 7).
For further analysis and determination of binding parameters, MST runs are combined into Analysis
Sets. Clicking the “Auto-Append” button will create a single Analysis Set which contains all loaded
MST runs as independent single runs. Alternatively, MST runs can be added by clicking the
symbol on the bottom right of each MST run thumbnail. This automatically creates a new Merge Set.
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To add replicate runs to a Merge Set, drag-and-drop them there directly. Merged runs will be
displayed with average values and error bars in the Dose Response Fit screen. The software allows
the merging of runs if the runs were collected using the same
- LED/excitation power - MST power - Capillary type - Acquisition mode (fluorescence channel) and optics module (optics contained in a Monolith
NT.Automated vs optics contained in a Monolith NT.115/NT.115Pico/NT.LabelFree)
Figure 2: Create Analysis and Merge Sets from MST runs in the Data Selection menu.
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Depending on the software used to perform the measurement, not all of these criteria may be saved
in the file. As a result, it may not be possible to merge data collected with different software. When
the user tries to add an incompatible run to a Merge Set, the software will reject the run and display
an incompatibility message. To create a custom number of different Merge and Analysis Sets, drag-
and-drop the MST run thumbnails.
Hint: Analysis Sets and Merge Sets can also be rearranged by simple dragging and
dropping.
Please note that names of Merge and Analysis Set can be edited for better description and
documentation by clicking the pen symbol on the respective flyout. The flyout appears upon mouse-
over in the respective Analysis Set field (see screenshot below). Also, after an Analysis Set was
created the analysis mode can still be switched between MST- and initial fluorescence analysis by
selecting the respective button. Another button allows to switch to the expert mode for analysis (see
next section for more details). Once Analysis Sets are created, binding data can be quantified.
7. Dose Response Fit
The Dose Response fit window allows for fitting MST data to obtain either dissociation constants
(Kds, using the law of mass action) or EC50 values (using the Hill equation). In the window, normalized
MST traces as well as corresponding dose response plots of the selected MST data are shown.
Figure 3 summarizes the data analysis and fitting workflow.
By selecting either an Analysis Set or a Merge Set on the left, the respective MST traces and their
dose response plots are displayed. By default, Fnorm-values in the dose response plot are calculated
from the ratio of normalized fluorescence F0/F1, where F0 corresponds to the normalized
fluorescence prior to MST activation. F1 is by default determined after an optimal MST power-
dependent time interval which yields the best signal-to-noise ratio.
Use the mouse or the arrow keys to navigate through the analysis tree in the left panel. The right
arrow key expands an Analysis Set or Merge Set, while the left arrow key collapses it.
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Data fitting is performed instantly after selecting the respective fit routine (Kd Model or Hill Model). Fitting requires initial values, which are determined automatically by the software (shown as Guess values in the fit model). Known parameters, such as target concentration, need to be fixed by checking the Fix checkbox. In some cases it may be required to guide the fitting algorithm by manually entering initial Guess values. Note: The Hill fit should only be used if the interaction involves a cooperative binding mode. A 1:1 interaction should always be fitted using the Kd Model. After a fit is performed, a range of statistical
parameters is automatically calculated and
displayed. For definitions, fit equations and
more information, click the button.
Replicates within one Merge Set are displayed
as average values and error bars representing
the standard deviation. Fits are applied to the
average values. In order to get an error
estimation on the resulting Kd, fit the replicates
individually and use this data to perform
statistics.
For in-depth data evaluation and fit refinement, single runs and MST traces can be highlighted either
by selection on the left, or by clicking on the respective MST trace or data point in the graphs. After
highlighting, outliers can be excluded from the fit (either greyed out or invisible ).
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Figure 3: Analysis of binding constants and EC50-values.
For a more in-depth evaluation of MST data, activate the Raw Data Inspection . Here,
effects such as sample aggregation, adsorption to capillary walls or fluorescence intensity variations
in the titration series can be easily identified. Please see the MO.Control software for more
information on sample quality and assay optimization.
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When preparing Merge Sets for presentation that contain non-binding negative control interactions, move the mouse cursor over the name of the Merge Set. A flyout will appear with a chain link symbol. Clicking this symbol initializes all enabled fit parameters as nonbinder, which means a horizontal line will be drawn through the points at their average value. This allows the comparison of nonbinders with binders in the Compare Results view. To revoke non-binder status, navigate to the data fitting section of the respective run and untick all unneeded checkboxes.
As an alternative to using the default analysis settings, the positions of F1 and F0 can be manually
adjusted after enabling the Expert Mode for the Analysis Set (see Figure 4). Using this mode, the F1
and F0 cursors can be placed anywhere along the MST timetraces. The Expert Mode should only be
used if the default analysis procedure did not yield satisfying results.
Figure 4: Activation of the Expert Mode and visualization of different cursor settings.
Similarly, when working with an Initial Fluorescence Analysis Set, the Expert Mode can be enabled
to analyze ligand-dependent photobleaching effects (bleaching rate). Please contact your
NanoTemper Technologies Support for more information.
Chart visuals: Chart colors can be changed in the Data Selection, Dose Response Fit and Compare
Results sections. All charts in the Dose Response Fit and Compare Results sections can be zoomed
and adjusted for optimal visualization. Use the Zoom Extent button to adjust all data in the chart to
the chart size. Zooming in-and-out of the chart is performed by scrolling the mouse wheel. Horizontal
or vertical zooming can be performed by pressing shift or control on the keyboard while scrolling,
respectively. Click and hold the mouse wheel and move the mouse to drag the chart (see Figure 5).
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Figure 5: Mouse control of chart visualization.
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8. Compare Results
The Compare Results tab allows for a side-by-side comparison of MST runs and Merge Sets within
an Analysis Set. In this tab, data and fitting results can also be exported in tabular and graphic
format, including all binding data and the algorithms used. By selecting an Analysis Set on the left,
all included data are plotted in the same chart. Selection of a Merge Set or a single MST run will
highlight the selected experiments and grey-out the remaining experiments in the Analysis Set.
Dropdown menus to change the Fit Model (Kd or Hill) and display type (Fnorm, Fnorm, Fraction Bound)
are located on the top of the dose response chart. While the Fraction Bound normalization is best
suited for a direct comparison of binding affinities, the Fnorm normalization provides additional
information about amplitude size and direction (please contact your NanoTemper Technologies
Customer Support for more information). Both charts and chart raw data can be exported as an
image file (.svg, .png or .pdf) or text file (.csv or .xlsx) for further use and external analysis.
In addition to the visualization options, the Compare Results menu also includes a table summarizing
number of averaged experiments (n), fit parameters, affinities and fit quality.
Finally, the Generate Full Report button summarizes all charts and tables into one single PDF. Click
the Generate Full Report button in the Dose Response Fit view to obtain a report even with unfitted
data.
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Contact
NanoTemper Technologies GmbH
Floessergasse 4
81369 Munich
Germany
Phone: +49 (0)89 4522895 0
Fax: +49 (0)89 4522895 60
http://www.nanotempertech.com
MicroScale Thermophoresis™ is a trademark.
NanoTemper® and Monolith® are registered trademarks.
NanoTemper® and Monolith® are registered in the U.S. Patent
and Trademark Office.
V10_2018-03-14
